Pre-mRNA introns play an important role in the regulation of gene expression for many eukaryotes because their presence allows for the occurrence of alternative splicing. Such alternative splicing results in the creation of multiple proteins from a single gene, many of which are expressed in cell- or tissue-specific patterns. The pre-mRNA introns are excised in a lariat conformation to produce mRNA. Following excision, the 3′ tails of the lariats are subject to exonucleolytic degradation up to the lariat branch point. The predominant pathway for further exonucleolytic degradation requires cleavage of the 2′-5′ bond located at the branch point. This cleavage event occurs via a RNA debranching enzyme, a 2′-5′ phosphodiesterase.
Although intron RNA sequences contain information necessary for their removal from pre-mRNAs, some introns contain additional information. In most eukaryotes microRNAs (miRNAs) and small nucleolar RNAs (snoRNAs) are encoded within introns. In studies with human cells it has been found that the vast majority of intronic miRNAs are excised from pre-mRNAs. Intronic snoRNAs, on the other hand, are processed from excised introns, as determined in baker's yeast, humans, and other eukaryotes.
Debranching and subsequent degradation of most intron RNAs are rapid, resulting in low steady state levels of intron RNAs relative levels of the corresponding mRNAs. The exceptions are intron sequences corresponding to RNAs with additional functions (e.g. snoRNAs). Studies in many different organisms have determined that cleavage of the 2′-5′ bond by an RNA debranching enzyme is important for the maturation of intron-encoded snoRNAs and mirtrons, which is another class of miRNAs that are processed from excised introns.
Genome-wide studies analyzing excised intron RNAs in fruit flies and yeast have identified new introns and alternative splicing patterns. These analyses relied on creating cell populations that accumulate excised intron RNAs at elevated levels due to either mutation of the gene encoding debranching enzyme or knock down of debranching enzyme expression with siRNA. Analysis of RNA samples with elevated levels of RNA lariats increases the detectability of rare splicing variants. Cells defective for RNA debranching activity accumulate excised introns in their lariat forms with shorted 3′ tails. Without the full length 3′ tail, information for the 3′ intron-exon junction is not obtainable from the intron lariat RNA sequences. However, studies have shown that the positions of RNA branch points may be deduced from analyzing intron RNA lariats. Direct information on branch points is only obtainable from analysis of RNA lariats. Therefore, there is a need to provide new compositions and methods for the analysis of RNA lariats that allow analysis of rare splicing variants and branch point sequences.